Nucleic acids encoding mutations in sodium channels related to epilepsy

ABSTRACT

An isolated nucleic acid molecule encoding a mutant alpha subunit of a mammalian voltage-gated sodium channel, wherein a mutation event selected from the group consisting of point mutations, deletions, insertions and rearrangements has occurred and said mutation event disrupts the functioning of an assembled sodium channel comprising this mutated subunit so as to produce an epilepsy phenotype, with the proviso that the mutation event is not a C2624T transition or a G4943A transition.

PRIORITY APPLICATION INFORMATION

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/451,126 filed Oct. 8, 2003 now U.S. Pat. No. 7,078,515,which corresponds to PCT Application No. PCT/AU01/01648, filed Dec. 20,2001, which claims the benefit of PR2203 filed Dec. 20, 2000. Thisapplication is also a continuation-in-part of U.S. patent applicationSer. No. 10/482,834 filed Oct. 12, 2004, which corresponds to PCTApplication No. PCT/AU02/00910 filed Jul. 8, 2002, which claims thebenefit of PS6452 filed Jul. 18, 2001, PSO910 filed Mar. 5, 2002, andPS2292 filed May 13, 2002. This application is also acontinuation-in-part of International Application No. PCT/AU2004/001051,filed Aug. 6, 2004, which claims the benefit of AU2003904154, filed Aug.7, 2003. The disclosure of U.S. patent application Ser. No. 10/451,126,U.S. patent application Ser. No. 10/482,834, and InternationalApplication No. PCT/AU2004/001051 are each incorporated herein byreference in their entireties.

TECHNICAL FIELD

The present invention relates to mutations in the α-subunit of mammalianvoltage-gated sodium channels which are associated with idiopathicepilepsies and other disorders such as myotonias and cardiacarrhythmias, and to polymorphisms in the gene encoding the α-subunit(SCN1A).

SEQUENCE LISTING PROVIDED ON CD-R

The Sequence Listing associated with the instant disclosure has beensubmitted as a 0.95 MB file on CD-R (in duplicate) instead of on paper.Each CD-R is marked in indelible ink to identify the Applicants, Title,File Name (Updated FP22576.ST25.txt), Creation Date (Oct. 31, 2005),Computer System (IBM-PC/MS-DOS/MS-Windows), and Docket No. (1386/13/2).The Sequence Listing submitted on CD-R is hereby incorporated byreference into the instant disclosure.

BACKGROUND ART

Generalised epilepsy with febrile seizures plus (GEFS+; MIM 604236) wasfirst described by Scheffer and Berkovic (1997) and is now recognised asa common epilepsy syndrome (Singh et al. 1999; Baulac et al. 1999;Moulard et al. 1999; Peiffer et al. 1999; Scheffer et al. 2000).Although GEFS+ is familial, it was initially difficult to recognise itas a distinct syndrome, because of clinical heterogeneity within eachfamily. The common phenotypes are typical febrile seizures (FS) andfebrile seizures plus (FS+); FS+ differs from FS in that the attackswith fever continue beyond age 6 years and/or include afebriletonic-clonic seizures. Less common phenotypes include FS+ associatedwith absences, myoclonic or atonic seizures, and even more-severesyndromes such as myoclonic-astatic epilepsy. That such phenotypicdiversity could be associated with the segregation of a mutation in asingle gene was established with the identification of a mutation in thevoltage gated sodium channel β1 subunit gene (SCN1B) (Wallace et al.1998). This mutation (C121W) changes a conserved cysteine residue,disrupting a putative disulfide bridge, which results in in vitro lossof function of the β1 subunit. Without a functional β1 subunit the rateof inactivation of sodium channel α-subunits decreases, which may causeincreased sodium influx, resulting in a more depolarised membranepotential and hyperexcitability. Modifier genes or the environment mayinteract with the SCN1B gene to account for clinical heterogeneity, butthe rarity of SCN1B mutations (Wallace et al. 1998) strongly suggestedadditional genes of large effect underlie GEFS+ in other families (Singhet al. 1999).

GEFS+ in four families has been mapped to chromosome 2q (Baulac et al.1999; Moulard et al. 1999; Peiffer et al. 1999; Lopes-Cendes et al.2000). Recently, mutations in the neuronal voltage gated sodium channelalpha-1 (SCN1A) subunit were described in two GEFS+ families (Escayg etal. 2000). The mutations (T875M and R1648H) are located in highlyconserved S4 transmembrane segments of the channel which are known tohave a role in channel gating. It was suggested that these mutations mayreduce the rate of inactivation of SCN1A and therefore have a similareffect as the β1-subunit mutation.

GEFS+ is clearly a common complex disorder, with a strong genetic basis,incomplete penetrance and genetic and phenotypic heterogeneity. Febrileseizures occur in 3% of the population, and thus this phenotype mayoccur sporadically in GEFS+ families, in addition to occurring as aresult of the GEFS+ gene (Wallace et al 1998). Also, although somefamilies segregate an autosomal dominant gene of major effect, in manycases clinical genetic evidence, such as bilineality, suggests that forsome small families the disorder is multifactorial (Singh et al 1999).Despite this, large families continue to be ascertained and withcritical phenotypic analysis, they provide opportunities to localise andultimately identify the genes involved.

DISCLOSURE OF THE INVENTION

As used herein, the terms “mutation”, “mutation event”, or “mutant” istaken to mean a change in the nucleotide sequence or amino acid sequencecomposition of a gene when compared to the corresponding wild-typesequence. In the context of the present invention, these terms mayinclude any change in the sequence composition, including polymorphismsand rare vartiations as disclosed herein, which give rise to an epilepsyphenotype.

The present inventors have identified new mutations in the alpha subunitof the voltage-gated sodium channel that are associated with epilepsy,in particular generalized epilepsy with febrile seizures plus (GEFS+).

According to one aspect of the present invention there is provided anisolated DNA molecule encoding a mutant alpha subunit of a mammalianvoltage-gated sodium channel, wherein a mutation event selected from thegroup consisting of point mutations, deletions, insertions andrearrangements has occurred and said mutation event disrupts thefunctioning of an assembled sodium channel so as to produce an epilepsyphenotype, with the proviso that the mutation event is not a C2624Ttransition or a G4943A transition and the DNA molecule does not have thenucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5.

Preferably said mutation event is a point mutation.

Typically the mutation event occurs in an intracellular loop, preferablyin the intracellular loop between transmembrane segments 2 and 3 ofdomain I, in the S4 segment of domain IV at amino acid position 1656, orin an S5 segment of a transmembrane domain. Preferably the mutationcreates a phenotype of generalised epilepsy with febrile seizures plus.

In one form of the invention the mutation is in exon 4 of SCN1A andresults in replacement of a highly conserved aspartic acid residue witha valine residue at amino acid position 188. The D188V mutation lies inthe intracellular loop just outside the S3 segment of domain I of SCN1Aand occurs as a result of an A to T nucleotide substitution at position563 of the SCN1A coding sequence as shown in SEQ ID NO:1.

In a further form of the invention the mutation is in exon 21 of SCN1Aand results in the replacement of a highly conserved valine residue witha leucine residue at amino acid position 1353. The V1353L mutation islocated in the S5 segment of domain III of SCN1A and occurs as a resultof a G to C nucleotide substitution at position 4057 of the SCN1A codingsequence as shown in SEQ ID NO:3.

In a still further form of the invention the mutation is in exon 26 ofSCN1A and results in the replacement of a highly conserved isoleucineresidue with a methionine residue at amino acid position 1656. TheI1656M mutation is located in the S4 segment of domain IV of SCN1A andoccurs as a result of a C to G nucleotide substitution at position 4968of the SCN1A coding sequence as shown in SEQ ID NO:5.

In addition, the polymorphisms identified in Table 3 (SEQ ID Numbers:7-9and 11) and the nucleic acid molecules recited in Table 4 (SEQ IDNOs:89-97, 108, 111-114, 136-144, and 153-154) form part of theinvention.

The present invention also encompasses DNA molecules in which one ormore additional mutation events selected from the group consisting ofpoint mutations, deletions, insertions and rearrangements have occurred.Any such DNA molecule will have the mutation associated with epilepsydescribed above and will be functional, but otherwise may varysignificantly from the DNA molecules set forth in SEQ ID NO:1, 3 and 5,Table 3 and Table 4.

The nucleotide sequences of the present invention can be engineeredusing methods accepted in the art for a variety of purposes. Theseinclude, but are not limited to, modification of the cloning,processing, and/or expression of the gene product. PCR reassembly ofgene fragments and the use of synthetic oligonucleotides allow theengineering of the nucleotide sequences of the present invention. Forexample, oligonucleotide-mediated site-directed mutagenesis canintroduce further mutations that create new restriction sites, alterexpression patterns and produce splice variants etc.

As a result of the degeneracy of the genetic code, a number ofpolynucleotide sequences, some that may have minimal similarity to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention includes each and every possiblevariation of a polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequences of the present invention, and all suchvariations are to be considered as being specifically disclosed.

The DNA molecules of this invention include cDNA, genomic DNA, syntheticforms, and mixed polymers, both sense and antisense strands, and may bechemically or biochemically modified, or may contain non-natural orderivatised nucleotide bases as will be appreciated by those skilled inthe art. Such modifications include labels, methylation, intercalators,alkylators and modified linkages. In some instances it may beadvantageous to produce nucleotide sequences possessing a substantiallydifferent codon usage than that of the polynucleotide sequences of thepresent invention. For example, codons may be selected to increase therate of expression of the peptide in a particular prokaryotic oreukaryotic host corresponding with the frequency that particular codonsare utilized by the host. Other reasons to alter the nucleotide sequencewithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringmutated sequence.

The invention also encompasses production of DNA sequences of thepresent invention entirely by synthetic chemistry. Synthetic sequencesmay be inserted into expression vectors and cell systems that containthe necessary elements for transcriptional and translational control ofthe inserted coding sequence in a suitable host. These elements mayinclude regulatory sequences, promoters, 5′ and 3′ untranslated regionsand specific initiation signals (such as an ATG initiation codon andKozak consensus sequence) which allow more efficient translation ofsequences encoding the polypeptides of the present invention. In caseswhere the complete coding sequence, including the initiation codon andupstream //regulatory sequences, are inserted into the appropriateexpression vector, additional control signals may not be needed.However, in cases where only coding sequence, or a fragment thereof, isinserted, exogenous translational control signals as described aboveshould be provided by the vector. Such signals may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers appropriate for the particularhost cell system used (Scharf et al., 1994).

The invention also includes nucleic acid molecules that are thecomplements of the sequences described herein.

According to still another aspect of the present invention there isprovided an isolated DNA molecule comprising the nucleotide sequence setforth in any one of SEQ ID NOs: 1, 7-9, 11, 89-97, 108, 111-114,136-144, and 153-154.

According to still another aspect of the present invention there isprovided an isolated DNA molecule consisting of the nucleotide sequenceset forth in any one of SEQ ID NOs: 1, 7-9, 11, 89-97, 108, 111-114,136-144, and 153-154.

The present invention allows for the preparation of purifiedpolypeptides or proteins from the polynucleotides of the presentinvention, or variants thereof. In order to do this, host cells may betransformed with a DNA molecule as described above. Typically said hostcells are transfected with an expression vector comprising a DNAmolecule according to the invention. A variety of expression vector/hostsystems may be utilized to contain and express sequences encodingpolypeptides of the invention. These include, but are not limited to,microorganisms such as bacteria transformed with plasmid or cosmid DNAexpression vectors; yeast transformed with yeast expression vectors;insect cell systems infected with viral expression vectors (e.g.,baculovirus); or mouse or other animal or human tissue cell systems.Mammalian cells can be used to express a protein using variousexpression vectors including plasmid, cosmid and viral systems such as avaccinia virus expression system. The invention is not limited by thehost cell employed.

The polynucleotide sequences, or variants thereof, of the presentinvention can be stably expressed in cell lines to allow long termproduction of recombinant proteins in mammalian systems. Sequencesencoding the polypeptides of the present invention can be transformedinto cell lines using expression vectors which may contain viral originsof replication and/or endogenous expression elements and a selectablemarker gene on the same or on a separate vector. The selectable markerconfers resistance to a selective agent, and its presence allows growthand recovery of cells which successfully express the introducedsequences. Resistant clones of stably transformed cells may bepropagated using tissue culture techniques appropriate to the cell type.

The protein produced by a transformed cell may be secreted or retainedintracellularly depending on the sequence and/or the vector used. Aswill be understood by those of skill in the art, expression vectorscontaining polynucleotides which encode a protein may be designed tocontain signal sequences which direct secretion of the protein through aprokaryotic or eukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, glycosylation,phosphorylation, and acylation. Post-translational cleavage of a“prepro” form of the protein may also be used to specify proteintargeting, folding, and/or activity. Different host cells havingspecific cellular machinery and characteristic mechanisms forpost-translational activities (e.g., CHO or HeLa cells), are availablefrom the American Type Culture Collection (ATCC) and may be chosen toensure the correct modification and processing of the foreign protein.

When large quantities of the gene are needed, such as for antibodyproduction, vectors which direct high levels of expression of thisprotein may be used, such as those containing the T5 or T7 induciblebacteriophage promoter. The present invention also includes the use ofthe expression systems described above in generating and isolatingfusion proteins which contain important functional domains of theprotein. These fusion proteins are used for binding, structural andfunctional studies as well as for the generation of appropriateantibodies.

In order to express and purify the protein as a fusion protein, theappropriate polynucleotide sequences of the present invention areinserted into a vector which contains a nucleotide sequence encodinganother peptide (for example, glutathionine succinyl transferase). Thefusion protein is expressed and recovered from prokaryotic or eukaryoticcells. The fusion protein can then be purified by affinitychromatography based upon the fusion vector sequence. The desiredprotein is then obtained by enzymatic cleavage of the fusion protein.

Fragments of polypeptides of the present invention may also be producedby direct peptide synthesis using solid-phase techniques. Automatedsynthesis may be achieved by using the ABI 431A Peptide Synthesizer(Perkin-Elmer). Various fragments of this protein may be synthesizedseparately and then combined to produce the full length molecule.

According to still another aspect of the present invention there isprovided an isolated polypeptide, said polypeptide being a mutant alphasubunit of a mammalian voltage-gated sodium channel, wherein a mutationevent selected from the group consisting of point mutations, deletions,insertions and rearrangements has occurred and said mutation eventdisrupts the functioning of an assembled sodium channel so as to producean epilepsy phenotype, with the proviso that said mutation event is nota T875M transition or a R1648H transition.

Preferably said mutation event occurs in an intracellular loop,preferably in the intracellular loop between transmembrane segments 2and 3 in domain I, in the S4 segment of domain IV at amino acid position1656, or in an S5 segment of a transmembrane domain of SCN1A. Preferablythe mutation creates a phenotype of generalised epilepsy with febrileseizures plus.

In one form of the invention the mutation event is a substitution inwhich a highly conserved aspartic acid residue is replaced with a valineresidue located in the intracellular domain located just outside the S3segment of domain I of SCN1A. Preferably the substitution is a D188Vtransition as illustrated in SEQ ID NO.:2.

In a further form of the invention the mutation event is a substitutionin which a highly conserved valine residue is replaced with a leucineresidue located in the S5 segment of domain III of SCN1A. Preferably thesubstitution is a V1353L transition as illustrated in SEQ ID NO:4.

In a still further form of the invention the mutation event is asubstitution in which a highly conserved isoleucine residue is replacedwith a methionine residue located in the S4 segment of domain IV ofSCN1A. Preferably the substitution is a I1656M transition as illustratedin SEQ ID NO:6.

In addition, the polymorphisms identified in Table 3 (SEQ ID Numbers:10and 12) and Table 4 (SEQ ID NOs: 119-127 and 165-173) form part of theinvention.

The isolated polypeptides of the present invention may have beensubjected to one or more mutation events selected from the groupconsisting of substitutions, deletions, insertions and rearrangements inaddition to the mutation associated with epilepsy. Typically thesemutation events are conservative substitutions.

According to still another aspect of the present invention there isprovided an isolated polypeptide comprising the sequence set forth inany one of SEQ ID NOs:2, 4, 6, 10, 12, 119-127 and 165-173.

According to still another aspect of the present invention there isprovided a polypeptide consisting of the amino acid sequence set forthin any one of SEQ ID NOs:2, 4, 6, 10, 12, 119-127 and 165-173.

According to still another aspect of the present invention there isprovided an isolated polypeptide complex, said polypeptide complex beingan assembled mammalian voltage-gated sodium channel, wherein a mutationevent selected from the group consisting of substitutions, deletions,insertions and rearrangements has occurred in the alpha subunit of thecomplex. Mutations include those in the intracellular loop betweentransmembrane segments 2 and 3, the S4 segment of domain IV at aminoacid position 1656, or in an S5 segment of a transmembrane domain of thealpha subunit. In a particular aspect an assembled mammalianvoltage-gated sodium channel bearing any such mutation in the alphasubunit will produce a phenotype of epilepsy, in particular generalisedepilepsy with febrile seizures plus, or other disorders associated withsodium channel dysfunction including, but not restricted to, myotoniassuch as hyperkalaemic periodic paralysis, paramyotonia congenita andpotassium aggravated myotonia, as well as cardiac arrhythmias such aslong QT syndrome.

In a particular aspect there is provided a complex, being an assembledmammalian voltage-gated sodium channel, bearing a mutation in theintracellular loop between transmembrane segments 2 and 3, the S4segment of domain IV at amino acid position 1656, or in an S5 segment ofa transmembrane domain of the SCN1A subunit of the channel.

According to still another aspect of the present invention there isprovided a method of preparing a polypeptide, said polypeptide being amutant alpha subunit of a mammalian voltage-gated sodium channel,comprising the steps of:

(1) culturing host cells transfected with an expression vectorcomprising a DNA molecule as described above under conditions effectivefor polypeptide production; and

(2) harvesting the mutant alpha subunit.

The mutant alpha subunit may also be allowed to assemble with othersubunits of the sodium channel, whereby the assembled mutant sodiumchannel is harvested.

Substantially purified protein or fragments thereof can then be used infurther biochemical analyses to establish secondary and tertiarystructure for example by X-ray crystallography of crystals of theproteins or by nuclear magnetic resonance (NMR). Determination ofstructure allows for the rational design of pharmaceuticals to interactwith the mutated sodium channel, alter the overall sodium channelprotein charge configuration or charge interaction with other proteins,or to alter its function in the cell.

It will be appreciated that, having identified mutations involved inepilepsy in these proteins, the mutant sodium channel alpha subunitswill be useful in further applications which include a variety ofhybridisation and immunological assays to screen for and detect thepresence of either a normal or mutated gene or gene product. Theinvention also enables therapeutic methods for the treatment of epilepsyand enables methods for the diagnosis of epilepsy. In particular theinvention enables treatment and diagnosis of generalised epilepsy withfebrile seizures plus, as well as other disorders associated with sodiumchannel dysfunction, as mentioned above.

Therapeutic Applications

According to one aspect of the invention there is provided a method oftreating epilepsy, in particular generalised epilepsy with febrileseizures plus, as well as other disorders associated with sodium channeldysfunction, including but not restricted to, myotonias such ashyperkalaemic periodic paralysis, paramyotonia congenita and potassiumaggravated myotonia, as well as cardiac arrhythmias such as long QTsyndrome, comprising administering a selective agonist, antagonist ormodulator of the sodium channel when a mutation event as described abovehas occurred, in particular, when it contains a mutation in theintracellular loop between transmembrane segments 2 and 3, in the S4segment of domain IV at amino acid position 1656, or in an S5 segment ofa transmembrane domain of an alpha subunit.

In still another aspect of the invention there is provided the use of aselective antagonist or modulator of the sodium channel when a mutationevent as described above has occurred, in particular, to a sodiumchannel when it contains a mutation in the intracellular loop betweentransmembrane segments 2 and 3, in the S4 segment of domain IV at aminoacid position 1656, or in an S5 segment of a transmembrane domain of analpha subunit, said mutation being causative of a disorder includingepilepsy, in particular generalised epilepsy with febrile seizures plusas well as other disorders associated with sodium channel dysfunction,including but not restricted to, myotonias such as hyperkalaemicperiodic paralysis, paramyotonia congenita and potassium aggravatedmyotonia, as well as cardiac arrhythmias such as long QT syndrome, inthe manufacture of a medicament for the treatment of the disorder.

In one aspect of the invention a suitable antagonist or modulator willrestore wild-type function to the sodium channels that contain amutation in an alpha subunit including those that form part of thisinvention.

Using methods well known in the art, a mutant sodium channel may be usedto produce antibodies specific for the mutant channel that is causativeof the disease or to screen libraries of pharmaceutical agents toidentify those that specifically bind the mutant sodium channel.

In one aspect, an antibody, which specifically binds to a mutant sodiumchannel, may be used directly as an antagonist or modulator, orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissues that express the mutant sodiumchannel.

In a still further aspect of the invention there is provided an antibodywhich is immunologically reactive with a polypeptide as described above,but not with a wild-type sodium channel or subunit thereof.

In particular, there is provided an antibody to an assembled sodiumchannel containing a mutation causative of a disorder as describedabove, in a subunit comprising the receptor. Such antibodies mayinclude, but are not limited to, polyclonal, monoclonal, chimeric, andsingle chain antibodies as would be understood by the person skilled inthe art.

For the production of antibodies, various hosts including rabbits, rats,goats, mice, humans, and others may be immunized by injection with apolypeptide as described or with any fragment or oligopeptide thereofwhich has immunogenic properties. Various adjuvants may be used toincrease immunological response and include, but are not limited to,Freund's, mineral gels such as aluminum hydroxide, and surface-activesubstances such as lysolecithin. Adjuvants used in humans include BCG(bacilli Calmette-Guerin) and Corynebacterium parvum.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to the mutant sodium channel have an amino acidsequence consisting of at least 5 amino acids, and, more preferably, ofat least 10 amino acids. It is also preferable that these oligopeptides,peptides, or fragments are identical to a portion of the amino acidsequence of the natural protein and contain the entire amino acidsequence of a small, naturally occurring molecule. Short stretches ofsodium channel amino acids may be fused with those of another protein,such as KLH, and antibodies to the chimeric molecule may be produced.

Monoclonal antibodies to a mutant sodium channel may be prepared usingany technique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique. (For example, see Kohler et al., 1975; Kozboret al., 1985; Cote et al., 1983; Cole et al., 1984).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature. (Forexample, see Orlandi et al., 1989; Winter et al., 1991).

Antibody fragments which contain specific binding sites for a mutantsodium channel may also be generated. For example, such fragmentsinclude, F(ab′)2 fragments produced by pepsin digestion of the antibodymolecule and Fab fragments generated by reducing the disulfide bridgesof the F(ab′)2 fragments. Alternatively, Fab expression libraries may beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity. (For example, see Huse et al.,1989).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between a sodium channel and its specific antibody. Atwo-site, monoclonal-based immunoassay utilizing antibodies reactive totwo non-interfering sodium channel epitopes is preferred, but acompetitive binding assay may also be employed.

In a further aspect, a suitable agonist may include a small moleculethat can restore wild-type activity of the sodium channel containingmutations in the alpha subunit as described above, or may include anantibody to a mutant sodium channel that is able to restore function toa normal level.

Small molecules suitable for therapeutic applications may be identifiedusing nucleic acids and peptides of the invention in drug screeningapplications as described below.

In a further aspect of the invention there is provided a method oftreating epilepsy, in particular generalised epilepsy with febrileseizures plus, as well as other disorders associated with sodium channeldysfunction, including but not restricted to, myotonias such ashyperkalaemic periodic paralysis, paramyotonia congenita and potassiumaggravated myotonia, as well as cardiac arrhythmias such as long QTsyndrome, comprising administering an isolated DNA molecule which is thecomplement (antisense) of any one of the DNA molecules described aboveand which encodes an RNA molecule that hybridizes with the mRNA encodinga mutant sodium channel alpha subunit, to a subject in need of suchtreatment.

Typically, a vector expressing the complement of the polynucleotides ofthe invention may be administered to a subject in need of suchtreatment. Antisense strategies may use a variety of approachesincluding the use of antisense oligonucleotides, injection of antisenseRNA, ribozymes, DNAzymes and transfection of antisense RNA expressionvectors. Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection, by liposomeinjections, or by polycationic amino polymers may be achieved usingmethods which are well known in the art. (For example, see Goldman etal., 1997).

In a still further aspect of the invention there is provided the use ofan isolated DNA molecule which is the complement of a DNA molecule ofthe invention and which encodes an RNA molecule that hybridizes with themRNA encoding a mutant sodium channel alpha subunit, in the manufactureof a medicament for the treatment of epilepsy, in particular generalisedepilepsy with febrile seizures plus, as well as other disordersassociated with sodium channel dysfunction, including but not restrictedto, myotonias such as hyperkalaemic periodic paralysis, paramyotoniacongenita and potassium aggravated myotonia, as well as cardiacarrhythmias such as long QT syndrome.

In further embodiments, any of the agonists, antagonists, modulators,antibodies, complementary sequences or vectors of the invention may beadministered alone or in combination with other appropriate therapeuticagents. Selection of the appropriate agents may be made by those skilledin the art, according to conventional pharmaceutical principles. Thecombination of therapeutic agents may act synergistically to effect thetreatment or prevention of the various disorders described above. Usingthis approach, therapeutic efficacy with lower dosages of each agent maybe possible, thus reducing the potential for adverse side effects.

Drug Screening

According to still another aspect of the invention, peptides of theinvention, particularly purified mutant sodium channel alpha subunitpolypeptide and cells expressing these, are useful for the screening ofcandidate pharmaceutical agents in a variety of techniques. It will beappreciated that therapeutic agents useful in the treatment of epilepsy,in particular generalised epilepsy with febrile seizures plus, as wellas other disorders associated with sodium channel dysfunction, includingbut not restricted to, myotonias such as hyperkalaemic periodicparalysis, paramyotonia congenita and potassium aggravated myotonia, aswell as cardiac arrhythmias such as long QT syndrome, are likely to showbinding affinity to the polypeptides of the invention.

Such techniques include, but are not limited to, utilising eukaryotic orprokaryotic host cells that are stably transformed with recombinantmolecules expressing the polypeptide or fragment, preferably incompetitive binding assays. Binding assays will measure for theformation of complexes between a mutated sodium channel alpha subunitpolypeptide or fragment and the agent being tested, or will measure thedegree to which an agent being tested will interfere with the formationof a complex between a mutated sodium channel alpha subunit polypeptideor fragment and a known ligand.

Another technique for drug screening provides high-throughput screeningfor compounds having suitable binding affinity to the mutant sodiumchannel alpha subunit polypeptides or sodium channels containing these(see PCT published application WO84/03564). In this stated technique,large numbers of small peptide test compounds can be synthesised on asolid substrate and can be assayed through mutant sodium channel ormutant sodium channel alpha subunit polypeptide binding and washing.Bound mutant sodium channel or mutant sodium channel alpha subunitpolypeptide is then detected by methods well known in the art. In avariation of this technique, purified polypeptides of the invention canbe coated directly onto plates to identify interacting test compounds.

The invention also contemplates the use of competition drug screeningassays in which neutralizing antibodies capable of specifically bindingthe mutant sodium channel compete with a test compound for bindingthereto. In this manner, the antibodies can be used to detect thepresence of any peptide that shares one or more antigenic determinantsof the mutant sodium channel.

The invention is particularly useful for screening compounds by usingthe polypeptides of the invention in transformed cells, transfected orinjected oocytes, or transgenic animals. A particular drug is added tothe cells in culture or administered to a transgenic animal containingthe mutant sodium channel and the effect on the current of the channelis compared to the current of a cell or animal containing the wild-typesodium channel. Drug candidates that alter the current to a more normallevel are useful for treating or preventing epilepsy, in particulargeneralised epilepsy with febrile seizures plus as well as otherdisorders associated with sodium channel dysfunction, as describedabove.

The polypeptides of the present invention may also be used for screeningcompounds developed as a result of combinatorial library technology.This provides a way to test a large number of different substances fortheir ability to modulate activity of a polypeptide. The use of peptidelibraries is preferred (see WO 97/02048) with such libraries and theiruse known in the art.

A substance identified as a modulator of polypeptide function may bepeptide or non-peptide in nature. Non-peptide “small molecules” areoften preferred for many in vivo pharmaceutical applications. Inaddition, a mimic or mimetic of the substance may be designed forpharmaceutical use. The design of mimetics based on a knownpharmaceutically active compound (“lead” compound) is a common approachto the development of novel pharmaceuticals. This is often desirablewhere the original active compound is difficult or expensive tosynthesise or where it provides an unsuitable method of administration.In the design of a mimetic, particular parts of the original activecompound that are important in determining the target property areidentified. These parts or residues constituting the active region ofthe compound are known as its pharmacophore. Once found, thepharmacophore structure is modelled according to its physical propertiesusing data from a range of sources including x-ray diffraction data andNMR. A template molecule is then selected onto which chemical groupswhich mimic the pharmacophore can be added. The selection can be madesuch that the mimetic is easy to synthesise, is likely to bepharmacologically acceptable, does not degrade in vivo and retains thebiological activity of the lead compound. Further optimisation ormodification can be carried out to select one or more final mimeticsuseful for in vivo or clinical testing.

It is also possible to isolate a target-specific antibody and then solveits crystal structure. In principle, this approach yields apharmacophore upon which subsequent drug design can be based asdescribed above. It may be possible to avoid protein crystallographyaltogether by generating anti-idiotypic antibodies (anti-ids) to afunctional, pharmacologically active antibody. As a mirror image of amirror image, the binding site of the anti-ids would be expected to bean analogue of the original receptor. The anti-id could then be used toisolate peptides from chemically or biologically produced peptide banks.

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

Diagnostic Applications

Polynucleotide sequences of the invention may be used for the diagnosisof epilepsy, in particular generalised epilepsy with febrile seizuresplus, as well as other disorders associated with sodium channeldysfunction, including but not restricted to, myotonias such ashyperkalaemic periodic paralysis, paramyotonia congenita and potassiumaggravated myotonia, as well as cardiac arrhythmias such as long QTsyndrome, and the use of the DNA molecules of the invention in diagnosisof these disorders, is therefore contemplated.

In another embodiment of the invention, the polynucleotides that may beused for diagnostic purposes include oligonucleotide sequences, genomicDNA and complementary RNA and DNA molecules. The polynucleotides may beused to detect and quantitate gene expression in biological samples.Genomic DNA used for the diagnosis may be obtained from body cells, suchas those present in the blood, tissue biopsy, surgical specimen, orautopsy material. The DNA may be isolated and used directly fordetection of a specific sequence or may be amplified by the polymerasechain reaction (PCR) prior to analysis. Similarly, RNA or cDNA may alsobe used, with or without PCR amplification. To detect a specific nucleicacid sequence, hybridisation using specific oligonucleotides,restriction enzyme digest and mapping, PCR mapping, RNAse protection,and various other methods may be employed. For instance directnucleotide sequencing of amplification products from the GABA receptorsubunits can be employed. Sequence of the sample amplicon is compared tothat of the wild-type amplicon to determine the presence (or absence) ofnucleotide differences.

According to a further aspect of the invention there is provided the useof a polypeptide as described above in the diagnosis of epilepsy, inparticular generalised epilepsy with febrile seizures plus, as well asother disorders associated with sodium channel dysfunction, as describedabove.

When a diagnostic assay is to be based upon mutant proteins constitutinga sodium channel, a variety of approaches are possible. For example,diagnosis can be achieved by monitoring differences in theelectrophoretic mobility of normal and mutant alpha subunit proteinsthat form part of the sodium channel. Such an approach will beparticularly useful in identifying mutants in which charge substitutionsare present, or in which insertions, deletions or substitutions haveresulted in a significant change in the electrophoretic migration of theresultant protein. Alternatively, diagnosis may be based upondifferences in the proteolytic cleavage patterns of normal and mutantproteins, differences in molar ratios of the various amino acidresidues, or by functional assays demonstrating altered function of thegene products.

In another aspect, antibodies that specifically bind mutant sodiumchannels may be used for the diagnosis of epilepsy, or in assays tomonitor patients being treated with agonists, antagonists, modulators orinhibitors of the mutant sodium channel. Antibodies useful fordiagnostic purposes may be prepared in the same manner as describedabove for therapeutics. Diagnostic assays to detect mutant sodiumchannels include methods that utilize the antibody and a label to detecta mutant sodium channel in human body fluids or in extracts of cells ortissues. The antibodies may be used with or without modification, andmay be labelled by covalent or non-covalent attachment of a reportermolecule.

A variety of protocols for measuring the presence of mutant sodiumchannels, including ELISAs, RIAs, and FACS, are known in the art andprovide a basis for diagnosing epilepsy, in particular generalisedepilepsy with febrile seizures plus, as well as other disordersassociated with sodium channel dysfunction, as described above. Theexpression of a mutant channel is established by combining body fluidsor cell extracts taken from test mammalian subjects, preferably human,with antibody to the channel under conditions suitable for complexformation. The amount of complex formation may be quantitated by variousmethods, preferably by photometric means. Antibodies specific for themutant channel will only bind to individuals expressing the said mutantchannel and not to individuals expressing only wild-type channels (ienormal individuals). This establishes the basis for diagnosing thedisease.

Once an individual has been diagnosed with the disorder, effectivetreatments can be initiated. These may include administering a selectivemodulator of the mutant channel or an antagonist to the mutant channelsuch as an antibody or mutant complement as described above. Alternativetreatments include the administering of a selective agonist or modulatorto the mutant channel so as to restore channel function to a normallevel.

Microarray

In further embodiments, complete cDNAs, oligonucleotides or longerfragments derived from any of the polynucleotide sequences describedherein may be used as probes in a microarray. The microarray can be usedto monitor the expression level of large numbers of genes simultaneouslyand to identify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, and to develop andmonitor the activities of therapeutic agents. Microarrays may beprepared, used, and analyzed using methods known in the art. (Forexample, see Schena et al., 1996; Heller et al., 1997).

According to a further aspect of the present invention, neurologicalmaterial obtained from animal models generated as a result of theidentification of specific sodium channel alpha subunit human mutations,particularly those disclosed in the present invention, can be used inmicroarray experiments. These experiments can be conducted to identifythe level of expression of specific sodium channel alpha subunits, orany cDNA clones from whole-brain libraries, in epileptic brain tissue asopposed to normal control brain tissue. Variations in the expressionlevel of genes, including sodium channel alpha subunits, between the twotissues indicates their involvement in the epileptic process either as acause or consequence of the original sodium channel mutation present inthe animal model. Microarrays may be prepared, as described above.

Transformed Hosts

The present invention also provides for the production of geneticallymodified (knock-out, knock-in and transgenic), non-human animal modelstransformed with the DNA molecules of the invention. These animals areuseful for the study of the function of a sodium channel, to study themechanisms of disease as related to a sodium channel, for the screeningof candidate pharmaceutical compounds, for the creation of explantedmammalian cell cultures which express a mutant sodium channel and forthe evaluation of potential therapeutic interventions.

Animal species which are suitable for use in the animal models of thepresent invention include, but are not limited to, rats, mice, hamsters,guinea pigs, rabbits, dogs, cats, goats, sheep, pigs, and non-humanprimates such as monkeys and chimpanzees. For initial studies,genetically modified mice and rats are highly desirable due to theirrelative ease of maintenance and shorter life spans. For certainstudies, transgenic yeast or invertebrates may be suitable and preferredbecause they allow for rapid screening and provide for much easierhandling. For longer term studies, non-human primates may be desired dueto their similarity with humans.

To create an animal model for a mutated sodium channel several methodscan be employed. These include but are not limited to generation of aspecific mutation in a homologous animal gene, insertion of a wild typehuman gene and/or a humanized animal gene by homologous recombination,insertion of a mutant (single or multiple) human gene as genomic orminigene cDNA constructs using wild type or mutant or artificialpromoter elements or insertion of artificially modified fragments of theendogenous gene by homologous recombination. The modifications includeinsertion of mutant stop codons, the deletion of DNA sequences, or theinclusion of recombination elements (lox p sites) recognized by enzymessuch as Cre recombinase.

To create a transgenic mouse, which is preferred, a mutant version of asodium channel alpha subunit can be inserted into a mouse germ lineusing standard techniques of oocyte microinjection or transfection ormicroinjection into embryonic stem cells. Alternatively, if it isdesired to inactivate or replace an endogenous sodium channel alphasubunit gene, homologous recombination using embryonic stem cells may beapplied.

For oocyte injection, one or more copies of the mutant sodium channelalpha subunit gene can be inserted into the pronucleus of ajust-fertilized mouse oocyte. This oocyte is then reimplanted into apseudo-pregnant foster mother. The liveborn mice can then be screenedfor integrants using analysis of tail DNA or DNA from other tissues forthe presence of the particular human subunit gene sequence. Thetransgene can be either a complete genomic sequence injected as a YAC,BAC, PAC or other chromosome DNA fragment, a complete cDNA with eitherthe natural promoter or a heterologous promoter, or a minigenecontaining all of the coding region and other elements found to benecessary for optimum expression.

According to still another aspect of the invention there is provided theuse of genetically modified non-human animals as described above for thescreening of candidate pharmaceutical compounds.

It will be clearly understood that, although a number of prior artpublications are referred to herein, this reference does not constitutean admission that any of these documents forms part of the commongeneral knowledge in the art, in Australia or in any other country.

Throughout this specification and the claims, the words “comprise”,“comprises” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred forms of the invention are described, by way of example only,with reference to the following examples and the accompanying drawings,in which:

FIG. 1. Generalised epilepsy with febrile seizures plus (GEFS+)pedigrees are shown for the three families. DNA was not available fromthose individuals not assigned a letter (X, Y, or Z) or a 0. A: Pedigreeof an Australian family with individual numbering for this family basedon FIG. 1 in Scheffer & Berkovic (1997). B: Pedigree of an Ashkenazifamily. C: Pedigree of a Druze family.

FIG. 2. Schematic of the alpha subunit of the sodium channel (SCN1A),showing the position of the three mutations identified in this study.

FIG. 3. Sodium channel amino acid alignments. Alignment of sodiumchannel amino acids surrounding the three SCN1A mutations.

FIG. 4 provides an example of ion channel subunit stoichiometry and theeffect of multiple versus single ion channel subunit mutations. FIG. 1A:A typical channel may have five subunits of three different types. FIG.1B: In outbred populations complex diseases such as idiopathicgeneralized epilepsies may be due to mutations in two (or more)different subunit genes. Because only one allele of each subunit gene isabnormal, half the expressed subunits will have the mutation. FIG. 1C:In inbred populations, both alleles of a single subunit gene will beaffected, so all expressed subunits will be mutated. FIG. 1D: Autosomaldominant disorders can be attributed to single ion channel subunitmutations that give rise to severe functional consequences.

FIG. 5 represents the location of mutations identified in the ionchannel subunits constituting the sodium channel. These examples includeboth novel and previously identified mutations.

FIG. 6 provides examples of epilepsy pedigrees where mutation profilesof ion channel subunits for individuals constituting the pedigree havebegun to be determined. These examples have been used to illustrate howthe identification of novel ion channel subunit mutations and variationsin IGE individuals can combine to give rise to the disorder.

MODES FOR PERFORMING THE INVENTION EXAMPLE 1 Clinical Diagnosis ofAffected Family Members

A group of 53 unrelated probands with GEFS+ phenotypes were studied.These subjects were ascertained on the basis of twin and family studiesand on the basis of routine clinical practice. Phenotypes in probandsand family members were classified as described elsewhere (Scheffer &Berkovic 1997; Singh et al 1999). Familial cases (n=36) were those inwhich at least one first-degree relative of the proband had a phenotypewithin the GEFS+ spectrum. Informed consent was obtained from allsubjects.

The Australian family in FIG. 1A, which has been described extensivelyelsewhere (Scheffer & Berkovic, 1997; Lopes-Cendes et al, 2000), is theoriginal pedigree leading to the initial delineation and description ofthe GEFS+ syndrome.

The Israeli family in FIG. 1B is of Ashkenazi origin and spans sixgenerations. Twelve family members had seizures. In the two oldestmembers (I-2, III-3) seizures had occurred in childhood but the datawere insufficient to allow classification of the phenotype. Of the 10other family members who had seizures, 3 had febrile seizures with onsetat age 9-13 months. All attacks occurred with fever and offset occurredbetween 1 and 4 years with 1 to 7 attacks each. Five had febrileseizures plus with onset at age 9-24 months, offset between 5 and 41years and 2 to 15 attacks each. Seizures during childhood were a mixtureof febrile seizures and afebrile tonic-clonic seizures, whereas therarely occurring seizures during teenage and adult years were allafebrile. Subject V-16 had a more severe phenotype with approximately 20febrile seizures at age 6 months to 5 years, 10 afebrile tonic-clonicseizures at age 5 to 15 years and occasional complex partial seizuresassociated with mild learning difficulties. She was classified as havingfebrile seizures plus and complex partial seizures. Her older sister(V-15) had typical febrile seizures plus, but their younger brother(V-17), aged 14 years, had no febrile seizures but had two afebriletonic-clonic seizures at ages 12 years 6 months and 14 years. Forpurposes of linkage analysis, he was regarded as affected, although hehad only afebrile tonic-clonic seizures. All affected subjects were ofnormal or superior intellect, except V-16 (see above) and all had anormal neurological examination. Electroencephalography (EEG) studieshad been performed infrequently during the active phase of the epilepsy,and the results usually either were normal or were reported to showgeneralised discharges.

The second Israeli family was of Druze origin; the parents were fromdifferent but proximate villages and were not known to be related. Thisfamily spans two generations, and four family members had seizures (FIG.1C). The proband aged 41 years (I-2) had had hundreds of tonic-clonicseizures, sometimes with fever. These began at age 4 years andcontinued, at a rate of approximately one per month, until the time ofthe study. The proband was mildly intellectually impaired. EEG showedgeneralized irregular spike-wave and polyspike-wave discharges, andfebrile seizures plus was diagnosed. Of her four children, the oldestwas unaffected (II-1), two had febrile seizures (II-2, II-4) and one hadfebrile seizures plus (II-3).

EXAMPLE 2 Isolation and Sequencing of SCN1A Genomic Clones

At the commencement of this study the full-length sequence of the humanSCN1A gene was not known. To determine this sequence a human BAC libraryobtained from Genome Systems was initially screened to identify humangenomic sequence clones containing the SCN1A gene. The BAC filters werescreened with a PCR product amplified with the primer pair 5′AGATGACCAGAGTGAATATGTGACTAC 3′ (SEQ ID NO:13) and 5′CCAATGGTAAAATAATAATGGCGT 3′ (SEQ ID NO:14) designed from the partialcDNA sequence of human SCN1A (Genbank Accession Number X65362).

The BAC filters were hybridised and washed according to manufacturersrecommendations. Initially, membranes were individually pre-hybridisedin large glass bottles for at least 2 hours in 20 ml of 6×SSC; 0.5% SDS;5× Denhardt's; 100 ug/ml denatured salmon sperm DNA at 65° C. Overnighthybridisations with [α-³²P]dCTP labelled probes were performed at 65° C.in 20 ml of a solution containing 6×SSC; 0.5% SDS; 100 ug/ml denaturedsalmon sperm DNA. Filters were washed sequentially in solutions of2×SSC; 0.5% SDS (room temperature 5 minutes), 2×SSC; 0.1% SDS (roomtemperature 15 minutes) and 0.1×SSC; 0.5% SDS (37° C. 1 hour if needed).

A number of BAC clones were identified from this hybridisation andBAC129e04 was selected for subcloning and sequencing. DNA from this BACclone was sheared by nebulisation (10 psi for 45 seconds). Sheared DNAwas then blunt ended using standard methodologies (Sambrook et al.,1989) and run on an agarose gel in order to isolate DNA in the 2-4 Kbsize range. These fragments were cleaned from the agarose using QIAquickcolumns (Qiagen), ligated into puc18 and used to transform competentXL-1 Blue E. coli cells. DNA was isolated from transformed clones andwas sequenced using vector specific primers on an ABI377 sequencer togenerate 1× coverage of the BAC clone. Sequence data were assembled incontigs using the Phred, Phrap and Gap4 high throughput sequencingsoftware. Exon-intron boundaries were predicted based on the rat Scn1aCDNA sequence (Genbank Accession Number M22253) due to the full lengthhuman cDNA sequence of SCN1A not being known.

The human SCN1A gene was determined to be 8,381 base pair in length andis organised into 27 exons spanning over 100 Kb of genomic DNA. Tofacilitate a comparison with related sodium channels SCN4A, SCN5A andSCN8A, the first untranslated exon of SCN1A is designated exon 1A andthe second exon, containing the start codon, remains exon 1 (Table 1).The SCN1A gene shows high homology to SCN2A and SCN3A at both the DNAand protein level. The close proximity of these genes to each other onchromosome 2 indicates likely duplication events during the evolution ofthe sodium channel gene family. Compared to SCN4A and SCN8A, additionalsequence is present in the 3′UTR of SCN1A, giving the final exon anoverall length of ˜3.3 Kb.

Inspection of the splice junctions of SCN1A shows that there is closeagreement with consensus splice motifs, with all introns bounded byGT-AG, except for two (introns 2 and 23). These introns exhibitdeviation from the consensus splice pattern and are bounded by AT-ACterminal dinucleotides. These rare splice site variations are conservedin other characterised sodium channel subunits (SCN4A, SCN8A and themore distantly related SCN5A), indicating their ancient origin.

The intron positions are also highly conserved between sodium channelsubunits, with most variation seen in the region that codes for thecytoplasmic loop between domains I and II of the gene (Table 1). Withinthis region, alternative splicing of exon 11 of SCN1A was found that wascomparable to the alternative splicing of exon 10B in SCN8A (Plummer etal. 1998). Cytoplasmic loop 1 varies in both length and composition andis the proposed site of functional diversity among different sodiumchannels (Plummer & Meisler, 1999).

EXAMPLE 3 Analysis of SCN1A for Mutations in Epilepsy

The determination of the genomic structure of SCN1A allowed the designof intronic primers (Table 2 and SEQ ID Numbers:15-88) to amplify eachof the 27 exons of SCN1A in order to test for mutations in patients withgeneralised epilepsy with febrile seizures plus (GEFS+). A total of 53unrelated patients (as described above) were screened by fluorescentsingle stranded conformation polymorphism (SSCP) analysis.

HEX-labelled primers were designed to amplify all exons of SCN1A (Table2). A 30 ng sample of patient DNA was amplified in a total volume of 10ul. Products were separated on non-denaturing 4% polyacrylamide gelscontaining 2% glycerol using the GelScan 2000 (Corbett Research). PCRproducts showing a conformational change were reamplified from 100 ng ofgenomic DNA with unlabelled primers and sequenced using the BigDyeTerminator ready reaction kit (Perkin Elmer) according to manufacturersinstructions.

A total of 53 unrelated patients with GEFS+ were screened by fluorescentSSCP, including two families consistent with mapping to the samelocation as SCN1A on chromosome 2 (FIGS. 1A and 1B). No mutations werefound in 17 sporadic cases of GEFS+ that were tested. Of the 36 familiestested, 3 were found to have point mutations in SCN1A, which alter theamino acid sequence and are not present in the control population(n=60). The phenotype in the family in FIG. 1A previously had beenmapped to chromosome 2 (Lopes-Cendes et al. 2000) and carries an A to Tmutation at position 563 of the SCN1A coding sequence. This mutationsegregates with affected family members. This mutation in exon 4 ofSCN1A results in a D188V amino acid substitution that lies just outsidethe S3 segment of domain I (FIG. 2). The aspartic acid residue isconserved in all identified sodium channels in humans as well as in manydifferent animal species, except the jellyfish which has an arginine atthis residue and the flatworm which has a serine (FIG. 3). The publishedrat Scn2a sequence (Genbank Accession Number NM_(—)012647) also has anarginine in place of the aspartic acid at residue 188.

A mutation in exon 21 (G to C nucleotide change at position 4057 of theSCN1A coding sequence) was found to segregate with GEFS+ in theAshkenazi family (FIG. 1B). This mutation changes a highly conservedamino acid (V1353L) located in the S5 segment of domain III (FIG. 2).One family member (V-13) did not carry the mutation (FIG. 1B). This wasdetermined by testing the DNA of a parent of this family member, sincethe subjects DNA was unavailable. This individual, who had typicalfebrile seizures that terminated at an early age, is likely to be aphenocopy. Mutations in the S5 segment of SCN4A that cause hyperkalemicperiodic paralysis have been shown also to affect the rate of channelinactivation (Bendahhou et al., 1999)

A third mutation (C to G nucleotide change at position 4968 of the SCN1Acoding sequence) discovered in the Druze family (FIG. 1C), changes anamino acid (I1656M) in the S4 segment of domain IV (FIG. 2). The S4segment has a role in channel gating and mutations in this region ofSCN1A reduce the rate of inactivation (Kuhn and Greef, 1996).

During the mutation screen of SCN1A several single nucleotidepolymorphisms (SNPs) were identified (Table 3). The R1928G variant wasfound at low frequency in both GEFS+ and control populations. The T1067Avariant was common in both populations and the remaining SNPs identifieddid not alter the amino acid sequence of SCN1A (Table 3).

EXAMPLE 4 Analysis of a Mutated Sodium Channels and Sodium Channel AlphaSubunits

The following methods are used to determine the structure and functionof mutated sodium channel or sodium channel alpha subunits.

Molecular Biological Studies

The ability of the mutated sodium channel as a whole or throughindividual alpha subunits to bind known and unknown proteins can beexamined. Procedures such as the yeast two-hybrid system are used todiscover and identify any functional partners. The principle behind theyeast two-hybrid procedure is that many eukaryotic transcriptionalactivators, including those in yeast, consist of two discrete modulardomains. The first is a DNA-binding domain that binds to a specificpromoter sequence and the second is an activation domain that directsthe RNA polymerase II complex to transcribe the gene downstream of theDNA binding site. Both domains are required for transcriptionalactivation as neither domain can activate transcription on its own. Inthe yeast two-hybrid procedure, the gene of interest or parts thereof(BAIT), is cloned in such a way that it is expressed as a fusion to apeptide that has a DNA binding domain. A second gene, or number ofgenes, such as those from a cDNA library (TARGET), is cloned so that itis expressed as a fusion to an activation domain. Interaction of theprotein of interest with its binding partner brings the DNA-bindingpeptide together with the activation domain and initiates transcriptionof the reporter genes. The first reporter gene will select for yeastcells that contain interacting proteins (this reporter is usually anutritional gene required for growth on selective media) The secondreporter is used for confirmation and while being expressed in responseto interacting proteins it is usually not required for growth.

The nature of the genes and proteins interacting with the mutant sodiumchannels can also be studied such that these partners can also betargets for drug discovery.

Structural Studies

Recombinant proteins corresponding to mutated sodium channel alphasubunits can be produced in bacterial, yeast, insect and/or mammaliancells and used in crystallographical and NMR studies. Together withmolecular modeling of the protein, structure-driven drug design can befacilitated.

EXAMPLE 5 Generation of Polyclonal Antibodies Against a Mutant SodiumChannel or Sodium Channel Alpha Subunit

Following the identification of new mutations in the alpha subunit ofthe sodium channel in individuals with generalised epilepsy with febrileseizures plus, antibodies can be made to the mutant channel which canselectively bind and distinguish mutant from normal protein. Antibodiesspecific for mutagenised epitopes are especially useful in cell cultureassays to screen for cells which have been treated with pharmaceuticalagents to evaluate the therapeutic potential of the agent.

To prepare polyclonal antibodies, short peptides can be designedhomologous to a sodium channel subunit amino acid sequence. Suchpeptides are typically 10 to 15 amino acids in length. These peptidesshould be designed in regions of least homology to other receptorsubunits and should also have poor homology to the mouse orthologue toavoid cross species interactions in further down-stream experiments suchas monoclonal antibody production. Synthetic peptides can then beconjugated to biotin (Sulfo-NHS-LC Biotin) using standard protocolssupplied with commercially available kits such as the PIERCE™ kit(PIERCE). Biotinylated peptides are subsequently complexed with avidinin solution and for each peptide complex, 2 rabbits are immunized with 4doses of antigen (200 ug per dose) in intervals of three weeks betweendoses. The initial dose is mixed with Freund's Complete adjuvant whilesubsequent doses are combined with Freund's Immuno-adjuvant. Aftercompletion of the immunization, rabbits are test bled and reactivity ofsera is assayed by dot blot with serial dilutions of the originalpeptides. If rabbits show significant reactivity compared withpre-immune sera, they are then sacrificed and the blood collected suchthat immune sera can be separated for further experiments.

This procedure is repeated to generate antibodies against wild-typeforms of receptor subunits. The antibodies specific for mutant sodiumchannels can subsequently be used to detect the presence and therelative level of the mutant forms in various tissues.

EXAMPLE 6 Generation of monoclonal Antibodies Against a Mutant SodiumChannel or Sodium Channel Alpha Subunit

Monoclonal antibodies can be prepared in the following manner.Immunogen, comprising intact mutated sodium channel or sodium channelalpha subunit peptides, is injected in Freund's adjuvant into mice witheach mouse receiving four injections of 10 ug to 100 ug of immunogen.After the fourth injection blood samples taken from the mice areexamined for the presence of antibody to the immunogen. Immune mice aresacrificed, their spleens removed and single cell suspensions areprepared (Harlow and Lane, 1988). The spleen cells serve as a source oflymphocytes, which are then fused with a permanently growing myelomapartner cell (Kohler and Milstein, 1975). Cells are plated at a densityof 2×10⁵ cells/well in 96 well plates and individual wells are examinedfor growth. These wells are then tested for the presence of sodiumchannel specific antibodies by ELISA or RIA using wild type or mutantsubunit target protein. Cells in positive wells are expanded andsubcloned to establish and confirm monoclonality. Clones with thedesired specificity are expanded and grown as ascites in mice followedby purification using affinity chromatography using Protein A Sepharose,ion-exchange chromatography or variations and combinations of thesetechniques.

TABLE 1 Comparison of Exon Sizes of SCN1A with Other Human SCNA Subunits

Note: D: Transmembrane domain; C: Cytoplasmic loop.

TABLE 2 Primer Sequences Used for Mutation Analysis of SCN1A Size ExonForward Primer Reverse Primer (bp) 1A TACCATAGAGTGAGGCGAGGATGGACTTCCTGCTCTGCCC 356 1 CCTCTAGCTCATGTTTCATGAC TGCAGTAGGCAATTAGCAGC448 2 CTAATTAAGAAGAGATCCAGTGACAG GCTATAAAGTGCTTACAGATCATGTAC 356 3CCCTGAATTTTGGCTAAGCTGCAG CTACATTAAGACACAGTTTCAAAATCC 263 4GGGCTACGTTTCATTTGTATG GCAACCTATTCTTAAAGCATAAGACTG 355 5AGGCTCTTTGTACCTACAGC CATGTAGGGTCCGTCTCATT 199 6 CACACGTGTTAAGTCTTCATAGTAGCCCCTCAAGTATTTATCCT 394 7 GAACCTGACCTTCCTGTTCTC GTTGGCTGTTATCTTCAGTTTC241 8 GACTAGGCAATATCATAGCATAG CTTTCTACTATATTATCATCCGG 320 9TTGAAAGTTGAAGCCACCAC CCACCTGCTCTTAGGTACTC 363 10 GCCATGCAAATACTTCAGCCCCACAACAGTGGTTGATTCAGTTG 480 11a TGAATGCTGAAATCTCCTTCTACCTCAGGTTGCTGTTGCGTCTC 306 11b GATAACGAGAGCCGTAGAGAT TCTGTAGAAACACTGGCTGG315 12 CATGAAATTCACTGTGTCACC CAGCTCTTGAATTAGACTGTC 347 13aATCCTTGGGAGGTTTAGAGT CATCACAACCAGGTTGACAAC 292 13b CTGGGACTGTTCTCCATATTGGCATGAAGGATGGTTGAAAG 277 14 CATTGTGGGAAAATAGCATAAGC GCTATGCAGAACCCTGATTG338 15a TGAGACGGTTAGGGCAGATC AGAAGTCATTCATGTGCCAGC 348 15bCTGCAAGATCGCCAGTGATTG ACATGTGCACAATGTGCAGG 276 16aGTGGTGTTTCCTTCTCATCAAG TCTGCTGTATGATTGGACATAC 387 16bCAACAGTCCTTCATTAGGAAAC ACCTTCCCACACCTATAGAATC 353 17CTTGGCAGGCAACTTATTACC CAAGCTGCACTCCAAATGAAAG 232 18TGGAAGCAGAGACACTTTATCTAC GTGCTGTATCACCTTTTCTTAATC 234 19CCTATTCCAATGAAATGTCATATG CAAGCTACCTTGAACAGAGAC 318 20CTACACATTGAATGATGATTCTGT GCTATATACAATACTTCAGGTTCT 216 21aACCAGAGATTACTAGGGGAAT CCATCGAGCAGTCTCATTTCT 303 21bACAACTGGTGACAGGTTTGAC CTGGGCTCATAAACTTGTACTAAC 297 22ACTGTCTTGGTCCAAAATCTG TTCGATTAATTTTACCACCTGATC 267 23AGCACCAGTGACATTTCCAAC GGCAGAGAAAACACTCCAAGG 272 24 GACACAGTTTTAACCAGTTTGTGTGAGACAAGCATGCAAGTT 207 25 CAGGGCCAATGACTACTTTGCCTGATTGCTGGGATGATCTTGAATC 477 26a CGCATGATTTCTTCACTGGTTGGGCGTAGATGAACATGACTAGG 247 26b TCCTGCGTTGTTTAACATCGGATTCCAACAGATGGGTTCCCA 288 26c TGGAAGCTCAGTTAAGGGAGAAGCGCAGCTGCAAACTGAGAT 261 26d CCGATGCAACTCAGTTCATGGAGTAGTGATTGGCTGATAGGAG 274 26e AGAGCGATTCATGGCTTCCAATCCTGCCTTCTTGCTCATGTTTTTCCACA 335 26f CCTATGACCGGGTGACAAAGCCTGCTGACAAGGGGTCACTGTCT 242 Note: Primer sequences are listed 5′ to 3′.Due to the large size of exons 11, 13, 15, 16, 21 and 26, the exons weresplit into two or more overlapping amplicons.

TABLE 3 SCN1A Polymorphisms Identified SCN1A polymorphism Frequency (%)Position Mutation Amino acid change GEFS+ Normal Intron 13 IVS13 − — 2.48.6 14 37C > A Exon 14 c.2522C > G — 2.4 8.6 Inron 15 IVS15 + 54A > G —36.3 23.6 Exon 15 c.2889T > C — 1.2 0.0 Exon 16 c.3199G > A T1067A 29.530.8 Exon 26 c.5782C > G R1928G 1.2 1.7 Note Total GEFS + samples = 53;Total normal sa

EXAMPLE 6 Identification of Mutations in Ion Channels

Human genomic sequence available from the Human Genome Project was usedto characterize the genomic organisation for each sodium channel subunitgene. Each gene was subsequently screened for sequence changes usingsingle strand conformation polymorphism (SSCP) analysis in a largesample of epileptics with common sporadic IGE subtypes eg juvenilemyoclonic epilepsy (JME), childhood absence epilepsy (CAE), juvenileabsence epilepsy (JAE) and epilepsy with generalized tonic-clonicseizures (TCS). Clinical observations can then be compared to themolecular defects characterized in order to establish the combinationsof mutant subunits involved in the various disease states, and thereforeto provide validated drug targets for each of these disease states. Thiswill provide a basis for novel drug treatments directed at the geneticdefects present in each patient.

The coding sequence for each of the ion channel subunits was alignedwith human genomic sequence present in available databases at theNational Centre for Biotechnology Information (NCBI). The BLASTNalgorithm was typically used for sequence alignment and resulted in thegenomic organisation (intron-exon structure) of each gene beingdetermined. Where genomic sequence for an ion channel subunit was notavailable, BACs or PACs containing the relevant ion channel subunit wereidentified through screening of high density filters containing theseclones and were subsequently sequenced.

Availability of entire genomic sequence for each ion channel subunitfacilitated the design of intronic primers spanning each exon. Theseprimers were used for both high throughput SSCP screening and direct DNAsequencing.

EXAMPLE 7 Sample Preparation for SSCP Screening

A large collection of individuals affected with epilepsy have undergonecareful clinical phenotyping and additional data regarding their familyhistory has been collated. Informed consent was obtained from eachindividual for blood collection and its use in subsequent experimentalprocedures. Clinical phenotypes incorporated classical IGE cases as wellas GEFS+ and febrile seizure cases.

DNA was extracted from collected blood using the QIAamp DNA Blood Maxikit (Qiagen) according to manufacturers specifications or throughprocedures adapted from Wyman and White (1980). Stock DNA samples werekept at a concentration of 1 ug/ul.

In preparation for SSCP analysis, samples to be screened were formattedinto 96-well plates at a concentration of 30 ng/ul. These master plateswere subsequently used to prepare exon specific PCR reactions in the96-well format.

EXAMPLE 8 Identification of Sequence Alterations in Ion Channel Genes

SSCP analysis of specific ion channel exons followed by sequencing ofSSCP bandshifts was performed on individuals constituting the 96-wellplates to identify sequence alterations.

Primers used for SSCP were labelled at their 5′ end with HEX and typicalPCR reactions were performed in a total volume of 10 μl. All PCRreactions contained 67 mM Tris-HCl (pH 8.8); 16.5 mM (NH₄)₂SO₄; 6.5 μMEDTA; 1.5 mM MgCl₂; 200 μM each DNTP; 10% DMSO; 0.17 mg/ml BSA; 10 mMβ-mercaptoethanol; 5 μg/ml each primer and 100 U/ml Taq DNA polymerase.PCR reactions were performed using 10 cycles of 94° C. for 30 seconds,60° C. for 30 seconds, and 72° C. for 30 seconds followed by 25 cyclesof 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 30seconds. A final extension reaction for 10 minutes at 72° C. followed.

Twenty μl of loading dye comprising 50% (v/v) formamide, 12.5 mM EDTAand 0.02% (w/v) bromophenol blue were added to completed reactions whichwere subsequently run on non-denaturing 4% polyacrylamide gels with across-linking ratio of 35:1 (acrylamide:bis-acrylamide) and containing2% glycerol. Gel thickness was 100 μm, width 168 mm and length 160 mm.Gels were run at 1200 volts and approximately 20 mA, at 22° C. andanalysed on the GelScan 2000 system (Corbett Research, Australia)according to manufacturers specifications.

PCR products showing a conformational change were subsequentlysequenced. This first involved re-amplification of the amplicon from therelevant individual (primers used in this instance did not contain 5′HEX labels) followed by purification of the PCR amplified templates forsequencing using QiaQuick PCR preps (Qiagen) based on manufacturersprocedures. The primers used to sequence the purified amplicons wereidentical to those used for the initial amplification step. For eachsequencing reaction, 25 ng of primer and 100 ng of purified PCR templatewere used. The BigDye sequencing kit (ABI) was used for all sequencingreactions according to the manufacturers specifications. The productswere run on an ABI 377 Sequencer and analysed using the EditViewprogram.

Table 4 shows the novel sequence changes identified in the ion channelsubunits to date.

EXAMPLE 9 Digenic Model Examples

In some instances a single mutation in an ion channel alone isinsufficient to give rise to an epilepsy phenotype. However combinationsof mutations each conferring a subtle change of function to an ionchannel, as proposed by the digenic model (PCT/AU01/00872), may besufficient to produce an epilepsy phenotype.

Using the mutations and variations in ion channel subunits that formpart of this invention, the digenic model may be validated through aparametric analysis of large families in which two abnormal allelesco-segregate by chance to identify mutations which act co-operatively togive an epilepsy phenotype. It is envisaged that the strategy of carefulclinical phenotyping in these large families, together with a linkageanalysis based on the digenic hypothesis will allow identification ofthe mutations in ion channels associated with IGEs. If molecular geneticstudies in IGE are successful using the digenic hypothesis, such anapproach might serve as a model for other disorders with complexinheritance.

The digenic hypothesis predicts that the closer the genetic relationshipbetween affected individuals, the more similar the sub-syndromes,consistent with published data (Italian League Against Epilepsy GeneticCollaborative Group, 1993). This is because more distant relatives areless likely to share the same combinations of mutated subunits.

Identical twins have the same pair of mutated subunits and the sameminor alleles so the sub-syndromes are identical. Affected sib-pairs,including dizygous twins, with the same sub-syndrome would also have thesame pair of mutated subunits, but differences in minor alleles wouldlead to less similarity than with monozygous twins. Some sib-pairs anddizygous twins, have quite different sub-syndromes; this would be due todifferent combinations of mutated subunits, when the parents have morethan two mutated alleles between them.

A special situation exists in inbred communities that parallelsobservations on autosomal recessive mouse models. Here the two mutatedalleles of the digenic model are the same and thus result in a trueautosomal recessive disorder. Because all affected individuals have thesame pair of mutated alleles, and a similar genetic background, thephenotypes are very similar.

In outbred communities approximately 1% of the population would have IGEgenotypes (2 mutated alleles) and 0.3% would clinically express IGE.Most of these would have mutations in two different channel subunits. Insuch communities most cases would appear “sporadic” as the risk to firstdegree relatives would be less than 10%.

For example, let there be three IGE loci (A,B,C) and let the frequencyof abnormal alleles (a*,b*,c*) at each locus be 0.027 and of normalalleles (a, b, c) be 0.973. Then, the distribution of genotypes aa*,a*a, a*a* and aa at locus A will be 0.0263 (0.027×0.973), 0.0263, 0.0007and 0.9467 respectively, and similarly for loci B and C. In thispopulation 0.8485 will have no mutated alleles (0.9467³), 0.1413 willhave one mutated allele (a* or b* or c*; 0.0263×0.9467²×6), 0.0098 willhave two abnormal alleles (0.0020 two same abnormal alleles, 0.0078, twodifferent abnormal alleles) and 0.00037 will have more than two abnormalalleles. Thus in this population 0.01, or 1%, will have two or moreabnormal alleles (IGE genotype), and the total abnormal allele frequencywill be 0.08 (3×0.027).

To determine the familial risks and allele patterns in affected pairs,the frequency distribution of population matings and the percentage ofchildren with 2 or more abnormal alleles must be determined. Thefrequency of matings with no abnormal alleles (0×0) is 0.72 (0.8485²),for 1×0 and 0×1 matings 0.24 (2×0.8485×0.1413), for a 1×1 mating 0.020,and for 2×0 and 0×2 matings 0.0166 etc. From this distribution ofmatings the frequency of children with 2 or more abnormal alleles can beshown to be 0.01. For example, the 0×2 and 2×0 matings contribute 0.0033of this 0.01 frequency (0.0166 [mating frequency]×0.2 [chance of thatmating producing a child with 2 or more abnormal alleles]).

To determine parental risk it can be shown that of children with 2abnormal alleles (IGE genotype), 0.49 derive from 1×1 matings where noparent is affected, 0.33 derive from a 2×0 and 0×2 matings etc. For the2×0 and 0×2 matings, half the parents have IGE genotypes and contribute0.16 (0.33/2) to the parental risk with the total parental risk of anIGE genotype being 0.258. The other matings that contribute to affectedparent-child pairs are 2×1, 1×2, 3×0, 0×3 etc.

The sibling risk of an IGE genotype is 0.305. For example 2×0 and 0×2matings contributed 0.08 to the sibling risk (0.33[fraction of childrenwith 2 abnormal alleles]×0.25[the chance of that mating producing achild with 2 or more abnormal alleles]). Similarly the offspring riskwas determined to be 0.248 by mating individuals with 2 abnormal alleleswith the general population. Thus at 30% penetrance the risk for IGEphenotype for parents of a proband is 0.077, for siblings 0.091, and foroffspring 0.074.

It can be shown that affected sib pairs share the same abnormal allelepair in 85% of cases. This is because of all affected sib pairs 44%derive from 1×1 matings and 23% from 0×2 and 2×0 matings where allaffected siblings have the same genotype. In contrast, 24% derive from1×2 matings and 9% from 3×1 and 2×2 matings etc where affected siblinggenotypes sometimes differ.

For affected parent-child pairs, genotypes are identical in only 58%. Ofaffected parent child pairs, 43% derive from 0×2 matings where gentoypesare identical, whereas 38% derive from 0×3 and 17% from 1×2 where themajority of crosses yield different affected genotypes.

Based on the digenic model it has been postulated that most classicalIGE and GEFS⁺ cases are due to the combination of two mutations inmulti-subunit ion channels. These are typically point mutationsresulting in a subtle change of function. The critical postulate is thattwo mutations, usually, but not exclusively, in different subunitalleles (“digenic model”), are required for clinical expression of IGE.

The hypothesis that similar phenotypes can be caused by the combinationof mutations in two (or more) different subunits (outbred communities),or by the same mutation in two (or more) alleles of the same subunit(inbred communities), may seem implausible. However, applying thedigenic hypothesis to the theoretical pentameric channel shown in FIG.1, in outbred communities IGE will be due to subunit combinations suchas α*αβ*βΔ, α*αββΔ* or ααβ*βΔ* (mutated subunits indicated by *). Ininbred communities α*α*ββΔ or ααβ*β*Δ combinations might cause IGEphenotypes. We assume that the mutations will not cause reducedexpression of the alleles and that the altered ion channel excitability,and consequent IGE phenotype, caused by mutations in two differentalleles is similar to that caused by the same mutation in both allelesof one subunit. Finally, subunit mutations with more severe functionalconsequences (eg breaking a disulphide bridge in SCN1B or amino acidsubstitution in the pore forming regions of SCN1A for GEFS⁺) causeautosomal dominant generalized epilepsies with a penetrance of 60-90%.Such “severe” mutations are rare (allele frequency <0.01%) and areinfrequent causes of GEFS⁺. They very rarely, or perhaps never, causeclassical IGE.

The relative separate segregation of classical IGE and GEFS⁺ phenotypesis an anecdotal clinical observation of ours (Singh et al., 1999),although the separation is not absolute. The separation is supported byprevious family and EEG studies of Doose and colleagues who described“type A” and “type B” liabilities which we may approximate the GEFS⁺ andclassical IGE groupings respectively (Doose and Baier, 1987).

The digenic model predicts that affected sib pairs will share the samegenes in 85% of cases whereas they will have at least one differentallele in the remaining 15%. In contrast, only 58% of parent-child pairsshare the same alleles in a 3 locus model. Thus there should be greatersimilarity of syndromes between sibling pairs than parent-child pairs.This would be most objectively measured by age of onset and seizuretypes.

Estimates for the risk of febrile seizures or IGE in relatives vary. Theestimates range from 5%-10% for siblings, 4%-6% for offspring, 3%-6% forparents, and 2-3% for grandparents. Underestimation may occur becauseIGE manifest in youth, and parents and particularly grandparents may beunaware of seizures in themselves in younger years. This is particularlytrue where there was stigma associated with epilepsy and where theepilepsy may have been mild and unrecognized. Underestimation of siblingand offspring risks occurs when unaffected young children are counted,some of whom will develop IGE in adolescence. Overestimation may occurwith misdiagnosis of seizures or inclusion of seizures unrelated to IGE(e.g. due to trauma or tumors)

In autosomal dominant models the risk to affected relatives reducesproportionally (50% for first degree relatives, 25% for second degreeetc). For all oligogenic or polygenic models the risk decreases morequickly. For a digenic model with three loci, the risks are 9.1% forsiblings, 7.4% for offspring, 7.7% for parents. Rigorous measurement ofthe familial recurrence rates, with careful phenotyping andage-corrected risk estimates could be compared with the predictions fromthe digenic model, and it is proposed to do this.

There is a small amount of information on IGE families regardinghaplotype distribution. For example, there is some evidence for a locuson 8q as determined by parametric linkage in a single family (Fong etal., 1998) and by non-parametric analysis in multiple small families(Zara et al., 1995). Interestingly, in the latter study the 8q haplotypenot infrequently came from the unaffected parent. This would be quitecompatible with the digenic model and evaluation of other data sets inthis manner could be used to test the hypothesis, and it is proposed todo this.

Following the analysis of one large family with epilepsy where the twomain phenotypes were childhood absence epilepsy (CAE) and febrileseizures (FS), the inheritance of FS was found to be autosomal: dominantand the penetrance 75%. However the inheritance of CAE in this familywas not simple Mendelian, but suggestive of complex inheritance with theinvolvement of more than one gene. The power of this large family wasused to explore the complex genetics of CAE further.

Linkage analysis on this family in which individuals with CAE, FS andFS+ were deemed affected led to the detection of linkage on chromosome5q and identification of a mutation in the GABRG2 gene (R43Q) which islocalised to this region (Wallace et al., 2001a; PCT/AU01/00729). All 10tested individuals with FS alone in this family had this mutation and 7CAE affected individuals in this family also had the mutation. To testthe digenic model of IGEs in the CAE affected individuals, the wholegenome screen of this family was reanalysed with only individuals withCAE considered affected. Linkage analysis was performed using FASTLINKv4.0, two-point lod scores were calculated assuming 50% penetrance and a2% phenocopy rate and individuals with FS or FS+ were coded as unknown.Markers producing a lod score greater than 1 were reanalysed without aphenocopy rate and at the observed penetrance for CAE in this family(30%). Results from the analysis revealed significant linkage tochromosome 14q22-q23 (lod 3.4). This provides strong evidence for asecond locus segregating with CAE affected individuals in this family.While the GABRG2 mutation is sufficient to cause FS, the CAE phenotypeis thought to be due to both the GABRG2 mutation and a mutationoccurring in a gene mapping to the 14q locus, as proposed by the digenicmodel.

For the application of the digenic model to sporadic cases of IGE andaffected individuals belonging to smaller families in which genotypingand linkage analysis is not a feasible approach to disease geneidentification, direct mutation analysis of ion channel genes in theseindividuals has been carried out as described above. In Table 4 there isprovided an indication of novel genetic alterations so far identifiedthrough mutation analysis screening of these individuals. FIG. 5provides an example to indicate where some of these mutations haveoccurred with respect to the sodium channel genes.

The identification of novel mutations and variations in ion channelsubunits in IGE individuals provides resources to further test thedigenic hypothesis and mutation profiles are starting to accumulate fora number of subunit changes that are observed in the same individuals.FIG. 6 provides results from some of these profiles.

FIG. 6A shows a 3 generation family in which individual III-1 hasmyoclonic astatic epilepsy and contains a N43del mutation in the SCN3Agene as well as an A1067T mutation in the SCN1A gene. Individual I-1also has the SCN3A mutation but alone this mutation is not sufficient tocause epilepsy in this individual. The SCN3A mutation has likely beeninherited from the grandfather through the mother, while the SCN1Amutation is likely to arise from the father. Both parents are unaffectedbut have yet to be screened for the presence of the mutations in thesesubunits. Individual II-1 is likely to contain an as yet unidentifiedion channel subunit mutation acting in co-operation with the SCN3Amutation already identified in this individual.

FIG. 6B is another 3 generation family in which individual III-1 hasmyoclonic astatic epilepsy due to a combination of the same SCN3A andSCN1A mutations as above. However, in this family both parents havefebrile seizures most likely due to the presence of just one of themutations in each parent, as proposed by the model. This is in contrastto individuals II-2 and II-3 in FIG. 6A who also contain one of themutations in these genes each. These individuals are phenotypicallynormal most likely due to incomplete penetrance of these mutations ineach case.

FIG. 6C shows a larger multi-generation family in which individual IV-5has a mutation in both the SCN3A and GABRG2 subunits. In combination,these give rise to severe myoclonic epilepsy of infancy but alone eithercause febrile seizures (GABRG2 mutation in III-3 and IV-4) or arewithout an effect (SCN3A mutation in III-2) as proposed by the model.

These examples therefore illustrate the digenic model as determined frommutation analysis studies of ion channel subunits in affectedindividuals and highlight the need to identify genetic alterations inthe genes encoding ion channel subunits.

TABLE 4 Examples of mutations and variations identified in sodiumchannel subunit genes Subunit Gene Exon/Intron DNA Mutation Amino AcidChange SEQ ID NOS SCN1A^(r) Exon 1 c111delC P37fsX91  89, 119 SCN1A^(ra)Exon 4 c563A→T D188V SCN1A^(r) Exon 9 c1342-c1352del I448X  90, 120SCN1A^(r) Exon 20 c3976G→C A1326P  91, 121 SCN1A^(ra) Exon 21 c4057G→CV1353L SCN1A^(r) Exon 24 c4556C→T P1519L  92, 122 SCN1A^(r) Exon 26c4905C→G F1635L  93, 123 SCN1A^(ra) Exon 26 c4968C→G I1656M SCN1A^(r)Exon 26 c5363-c5364ins N1788fsX1796  94, 124 SCN1A^(r) Exon 26 c5536-S1846fsX1856  95, 125 c5539delAAAC SCN1A^(r) Exon 26 c5643G→C E1881D 96, 126 SCN1A^(r) Exon 26 c5870A→G E1957G  97, 127 SCN8A^(r) Exon 14c3148G→A G1050S  98, 128 SCN1B^(ra) Exon 3 c253C→T R85C SCN1B^(ra) Exon3 c363C→G C121W SCN1B^(r) Exon 3 c367G→A V123I  99, 129 SCN1B^(r) Exon 3c373C→T R125C 100, 130 SCN2A^(r) Exon 21 c3988C→T L1330F 101, 131SCN2A^(r) Exon 25 c4687C→G L1563V 102, 132 SCN2A^(r) Exon 26 c5465C→TA1822V 103, 133 SCN1A^(ca) Exon 16 c3199A→G T1067A SCN1A^(ca) Exon 26c5782C→G R1928G SCN8A^(c) Exon 14 c3076C→T R1026C 104, 134 SCN3A^(c)Exon 1 c127-129delAAT N43del 105, 135 SCN1A^(r) Exon 15 c2889T→C —SCN3A^(r) Exon 13 c1971G→A — 106 SCN3A^(r) Exon 27 c5511C→T — 107SCN1A^(c) Exon 14 c2522C→G — SCN1A^(c) Exon 26 c5418G→A — 108 SCN3A^(c)Exon 13 c1884T→A — 109 SCN3B^(c) Exon 3 c438C→T — 110 SCN1A^(r) Intron 8IVS8(−9-10)delTT — 111 SCN1A^(r) Intron 10 IVS10-47T→G — 112 SCN1A^(r)Intron 18 IVS18 + 1G→A — 113 SCN1A^(r) Intron 22 IVS22 − 14T→G — 114SCN8A^(r) Intron 6 IVS6 + 9C→T — 115 SCN3A^(r) Intron 23 IVS23-31delT —116 SCN8A^(c) Intron 15 IVS15 + 20G→A — 117 SCN1B Intron 1 IVS1 + 15G→T118 SCN1A Exon 5 c664C→T R222X 136, 165 SCN1A Exon 8 c1152G→A W384X 137,166 SCN1A Exon 9 c1183G→C A395P 138, 167 SCN1A Exon 9 c1207T→C F403L139, 168 SCN1A Exon 9 c1237T→A Y413N 140, 169 SCN1A Exon 9 c1265T→AV422E 141, 170 SCN1A Exon 21 c4219C→T R1407X 142, 171 SCN1A Exon 26c5339T→C M1780T 143, 172 SCN1A Exon 26 c5674C→T R1892X 144, 173 SCN1BExon 3 c254G→A R85H 145, 174 SCN2A Exon 6A c668G→A R223Q 146, 175 SCN2AExon 16 c2674G→A V892I 147, 176 SCN2A Exon 17 c3007C→A L1003I 148, 177SCN2A Exon 19 c3598A→G T1200A 149, 178 SCN2A Exon 20 c3956G→A R1319Q150, 179 SCN2A Exon 12 c1785T→C 151 SCN2A Exon 27 c4919T→A 152 SCN1AIntron 9 IVS9 − 1G→A 153 SCN1A Intron 23 IVS23 + 33G→A 154 SCN2A Intron7 IVS7 + 61T→A 155 SCN2A Intron 19 IVS19 − 55A→G 156 SCN2A Intron 22IVS22 − 31A→G 157 SCN2A Intron 2 IVS2 − 28G→A 158 SCN2A Intron 8 IVS8 −3T→C 159 SCN2A Intron 11 IVS11 + 49A→G 160 SCN2A Intron 11 IVS11 − 16C→T161 SCN2A Intron 17 IVS17 − 71C→T 162 SCN2A Intron 17 IVS17 − 74delG 163SCN2A Intron 17 IVS17 − 74insG 164

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1. An isolated DNA comprising the sequence of SEQ ID NO:
 101. 2. Anexpression vector comprising an isolated DNA molecule as claimed inclaim
 1. 3. An isolated cell transformed with an isolated DNA moleculeas claimed in claim
 1. 4. The isolated cell of claim 3, wherein the cellis an eukaryotic cell.
 5. The isolated cell of claim 3, wherein the cellis a bacterial cell.
 6. A method of preparing a polypeptide comprisingthe steps of: (1) culturing cells as claimed in claim 3 under conditionseffective for polypeptide production; and (2) harvesting the polypeptideencoded by SEQ ID NO:101.
 7. An isolated DNA comprising the sequence ofSEQ ID NO:
 142. 8. An expression vector comprising an isolated DNAmolecule as claimed in claim
 7. 9. An isolated cell transformed with anisolated DNA molecule as claimed in claim
 7. 10. The isolated cell ofclaim 9, wherein the cell is an eukaryotic cell.
 11. The isolated cellof claim 9, wherein the cell is a bacterial cell.
 12. A method ofpreparing a polypeptide comprising the steps of: (1) culturing cells asclaimed in claim 9 under conditions effective for polypeptideproduction; and (2) harvesting the polypeptide encoded by SEQ ID NO:142.